Undecapeptides



r 3,258,502 Ce Patented August 23, 1966 3,268,502 UNDECAPEPTIDES Klaus Liiblre, Eberhard Schriider and Reinhard Helnpel,

Berlin, Germany, assiguors to Schering AG, Berlin, Germany No Drawing. Filed Dec. 23, 1964, Ser. No. 420,788 Claims priority, application Germany, Jan. 9, 1964,

Sch 34,444

7 Claims. (Cl. 260-1125) The present invention relates to new undecapeptides and to methods of producing the same, and more particularly to undecapeptides having blood pressure lowering activity, smooth muscles stimulating activity and other important activities.

It is known that there is present in the salivary glands of marine animals which manufacture and can discharge inks, such as sepia, cuttlefish, squid and the octopus, for example eledone moschat a, contain the undecapeptide eledoisin, the structure of which was clarified by Ersparmer and Anastasi in Experientia 18 (1962), page 58. The structural formula of eledoisin is as follows:

L PYROGLUTAMYL L PROLYL L SERYL L- LYSYL L ASPARAGYL-L-ALANYL-L-PHENYL- ALANYL L-ISOLEUCYL-GLYCYL-L-METHION- INAMIDE A confirmation of this structure was provided by the synthesis of Sandrin and Boissonnas in Experienta 18 (1962), page 59.

Pharmacological testing on human beings resulted in the conclusion that the peptide activity provides clinical and therapeutic value as highly active blood pressure lowering, blood vessel dilating and breathing stimulating agents. This was set forth by Sicuteri, Fancuillacci, Franchi and Michelacci in Experientia 19 (1963), page 44.

It is a primary object of the present invention to provide new undecapeptides which are somewhat analogous in structure to eledoisin, but which are much cheaper and easier to produce and yet have at least equal the activity of eledoisin.

It is another object of the present invention to provide methods of producing the new polypeptides of this invention.

It is yet another object of the present invent-ion to provide new undecapeptides which have a highly effective blood pressure lowering action.

It is a further object of the present invention to provide for the reduction of blood pressure of patients requiring the same by the administration of the new undecapeptides of this invention.

Other objects and advantages of the present invention will be apparent from a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainly comprises an undecapeptide of the formula:

L-pyroglutamyl-L-prolyl-L-seryl-L-lysyl-R -L-alanyl- L-phenylalanyl-R -glycyl-L-leucyl-L-methioninamide wherein R is selected from the group consisting of L-glutamyl, L-glutaminyl, L-asparaginyl and glycyl and wherein R is selected from the group consisting of L-isoleucyl, L-leucyl and Lvalyl, and further wherein R in the case that R is L-leucyl or L-valyl can also be L-asparagyl.

The synthesis of the new compounds of the present invention can be carried out by the usual method used in peptide coupling, for example as set forth in the monograph of Greenstein and Winitz in Chemistry of the Amino Acids, I. Wiley & Sons, New York, London (1961), preferably by the anhydride-, the azide-, the carbodiimideor the activated ester-method. The amino acid sequence is advantageously built up from small peptide units, for example as shown in Table 1 and in Table 2 below. If necessary, the functional groups which are not concerned with the condensation can intermediately be protected, for example by reductive or hydrolytically easily splittable radicals.

The end products themselves are preferably obtained by coupling a heptapeptide and a tetrapeptide (note Table 1). The C-terminal carboxyl group of the heptapeptide thus exists first in the form of the methyl ester which is saponified to free the carboxyl group. The saponification can be carried out in normal manner in alkaline medium. It is particularly advantageous to free the Cterminal carboxyl group by enzymatic saponification, preferably with chymotrypsin or trypsin.

By this enzymatic process which is for the first time used in the synthesis of peptides on the one hand avoids the racemization which is necessary in the case of the usual alkaline saponification, which in the instant case is particularly difiicult to accomplish because the treatment is of a long peptide, and on the other hand, is advantageous as compared to ordinary alkaline saponification in the, selectivity of the reaction which is guaranteed in the case of the enzymatic saponification.

The subsequent coupling with the C-terminal tetrapeptideamide (Table 1) ultimately proceeds to the fully protected active agent in which the disturbing functional groups are block by easily splittable radicals (the e-amino groups of the lysine by canbo -tert.-butoxy-groups; the y-carboxyl groups of the glutamic acid by the tert.-butyl group). The splitting off of the protective groups following usual procedures finally gives the desired undecapeptide.

TABLE 1 B00 OtBu Pyroglu Pro Ser Lys OMo Cho Glu Ala Phe OMo BCI C (IJtBu Pyroglu Pro Ser Lys {-OH H Glu Ala Phe Ol\ie 1|3OG (|)tBu I Pyroglu Pro Se! Lys Glu Ala Phe -OMe IIZOC (|)t]3u (Pyroglu Pro Ser Lys Glu Ala P110 OII II- Ilou Gly Lou Mot NH;

]|3OO ()tBu Pyroglu Pro Ser Lys Glu Ala Phe Ileu Gly Lou Met NH (VII) Pyroglu Pro Ser Lys Glu Ala Pho Ileu Gly Leu Mot NI-I;

(VIII) TABLE 2 ..[@NHNH II- Ileu Gly Lou Met -NH1 BOC| Gly Ala Plie Ileu Gly Leu Met NH,

Bfi 0 Pyroglu Pro Ser Lys OII 1-I Gly Ala Phe Ilou Gly Leu Met ]NH;

1300 l Pyroglu Pro Ser Lys Gly Ala Phe Ileu Gly Leu Met NH;

, Pyroglu Pro Ser Lys Gly Ala Phe Ileu Gly Leu Met |NH,

The production of the new compounds of the present invention provides considerable preparation advantages as compared to the synthesis of eledoisin, which results from the choice of the substituted amino acids. Thus, there is a decreased tendency of the intermediate products to enter into side reactions in the synthesis of the glutamyl -analogs. Thus, for example, glutamic acid exhibits much less of a tendency to form glutarimides than does the original aspartic acid to form succinimide.

A further advantage exists in the exchange of the isoleucine radical in position B of the eledoisin by value or leucine, since pure isoleucine is considerably more diflicult to obtain than either of the other two amino acids.

All of the eledoisin analogous compounds of the pres- (guinea pig ileum) and on the blood pressure (rabbits) the same or practically the same activity as eledoisin. Only the Len -analog exhibits a somewhat lower activity,

r which however still exists in the same order of magnitude as, for example, the action of bradykinin (note Table 3 below).

From the pharmacological standpoint, the Glu -analog exhibits the advantage of greater solubility. Cons 7O quently it is possible to use higher doses, for example ent invention exhibit in their action on the smooth muscles 75 for the physiological building of the active agent.

TABLE 3 Blood pressure lowering in percent on rabbits Relative activity calculated with respect to- Dose/kg. Number oi Tests mg 1 mg 2 mg. 5 mg. mg. mg. 50 mg. Eledoisin, Bradykinin,

percent percent Giu -E1cdoisin 13 17 22 24 29 32 37 3 100 1, 000 Asp(NH Elcdoisin 10 24 28 27 31 32 38 5 100 1, 000 Asp(NHQ VaN-Eledoisim 8 13 18 17 27 29 33 4 100 1, 000 Gly -Eledoisin 16 19 22 25 3O 35 4 100 1, 000 Gly Leu EledoisiiL None 2 12 4 10 100 Leu -Elcdoisin 1. None 8 14 3 10 100 (a) Eledoisin 3 8 19 27 30 33 38 (b) Bradykinin None 7 20 The following examples are given to further illustrate the present invention. The scope of the invention is not, however, meant to be limited to the specific details of the examples.

EXAMPLE 1 (a) Carbobenzoxy-L-glutamyl-v-terL-butyl ester-L-alanyl- L-phenylalanine-methyl ester (1) 10.5 g. of carbobenzoxy-Lglutamic acid-'y-tert-butyl ester-a-hydrazide [Ea Klieger and H. Gibian, Liebigs Ann. Chem. 655, 195 (1962)], are suspended in 39.6 cc. of 1.5% solution of hydrochloric acid in tetrahydrofurane at 20 C. and reacted with 3.65 cc. of tert.-butyl nitrite in 20 cc. of tetrahydrofurane. After everything has gone into solution, it is diluted with 300 cc. of ethyl acetate, shaken at the lowest possible temperature with a saturated sodium bicarbonate solution and dried over sodium sulfate. The azide solution is reacted with a mixture of 10 g. of L-alanyl-L-phenylalanine-methyl ester-hydrochloride and 4.9 cc. of triethylamine in cc. of dimethyl formamide. After the usual working up 12.3 g. of the Compound 1 above is obtained. The melting point is 142-145 C. (from ethyl acetate); x] =26.6 (c.:l, methanol).

(b) L-gluzamyl-y-terL-butyI cster-L-alanyl-L-phenylaIanine-melhyl ester (H) Compound 11 is obtained from 10 g. of the carbobenzoxy compound by catalytic hydrogenation in the presence of palladium black in methanol. The yield is 7.75 g. oil; [a] 12.3 (0. 1, methanol).

(6) L-pyroglutamyZ-L-prolyl-L-seryl-e-terL-butyl0xycarbonyl-L-lysine (IV) 15.0 g. of L-pyroglutamyl-L-prolyl-Lseryl-e-tert.-butyl' oxycarbonyl-Llysine-methyl ester (111) [Ed Sandrin and R. A. Boissonnas, Experientia XVIII, 77 (1962] are dissolved in the smallest possible amount of water and reacted with 30 cc. of 1 n KOH. After 3 hours of standing at room temperature, the mixture is introduced into a column over Dowex-SO (H+-form) and washed with water. The solution is freeze-dried. The yield is 14.6 g., [a] =l11(c.:1,water).

(ll) L pyroglutamyl-L-prolyl-L-seryle-tert.-butyloxycarbonyl-L-lysyI-L-glutamyl-y-lerL-butylester L-alanyl-L- phenylzilanine-methyl ester (V) 2 g. of L-pyroglutamyl-L-prolyl-L-seryl-s'tert-butyloxycarbonyl-L-lysine and 1.61 g. of tripeptide ester are dissolved in 20 cc. of dimethyl formamide and reacted at 15 C. with a solution of 0.84 g. of N,N'-dicyclohexylcarbodiimide in dimethyl forrnamide. After 2 days at 0 C., the precipitated dicyclohexyl urea is filtered off under suction and concentrated under vacuum. The residue is taken up in chloroform, shaken out in normal manner and concentrated. After precipitation from ethanol/ petroleum ether there is obtained 2.4 g. of compound V. The melting point is 165167 C., [a] 52.0 (c.=1, methanol).

The same compound can be obtained, for example, from L-pyroglutamyl-L-prolyl-L-seryl-e-tert-butyloxycarbonyl-L-lysine-hydr-azide [Ed Sandrin and R. A. Boissonnas, Experientia XVIII, 77 (1962)] using the azide method.

(8) L pyroglulamyl-L-pr0lyl-Lseryl-e-terf.-butyl0xycarbonyl L-lysyl-L-glutamyl-y-terL-bmylest'er-L-alanyl-L- phenylalanine (VI) 2.4 g. of the methyl ester of (V) are enzymatically saponified with chymotrypsin (molar enzyme-substrate ratio 1:2000) in dimethyl formamide/ acetate buffer (pH 7.0 maintained constant with 0.5 n ammonia by means of an autotritator). The solution is heated to 60 C. for working up, filtered off under suction from the denatured enzyme and after acidification with hydrogen chloride under cooling, the peptide-acid is taken up in chloroform. The solution is washed until neutral and dried over sodium sulfate. The yield is 1.4 g., [011 41.8 (c.=1, methanol).

(f) L pyroglutamyl-L-prolyl-L-se1yl-e-tehfi-butyloacycnrbonyl-L-lysyl-L-glutamyl y-terL-buty[ester L-a'lanyl-L- phanylalanyl-L-isoleucyl-glycyl-bZeucyl-L methioninamide (VII) 1.36 g. of the N-terminal heptapeptide acid (VI) are dissolved in dimethyl formamide and therewith mixed with a mixture of 1.3 g. of L-isoleucyl-gjlycyl-L-leucyl- L-methioninamide-hydrochloride and 0.42 cc. of triethylamine in dimethyl formarnide from which the triethylammonium chloride has been filtered off. 1.03 g. of N,N'-dicyclo hexylcarbodiimide, also dissolved in dimethyl formam-ide, are added at -15 C. and the reaction mixture is allowed to stand for 2 days at 0 C. After suction filtration of the dicyclohexyl urea, the remaining solution is concentrated under vacuum and the residue is rubbed with citric acid solution, water, saturated sodium bicarbonate solution and water. The yield is 1.08 g. (g) L pyroglutamyl L prolyl L seryl L-lysyl-L- glutamyl L alanyl L phenylwlanyl L isoleucylglycyl L leucyl L methioninamide, glu eledoisin (VIII) Hydrogen chloride is introduced at 0 C. for 1 hour into a solution of 0.9 g. of the fully protected undecapeptide in glacial acetic acid. After standing for 1 hour at 0 C. and an additional hour at room temperature it is precipitated with ether. The yield is 0.75 g. For purification it is subjected to chromatography on a SE- Seph-adex column (75% methianolic collidine acetate buffer, pH 5.5, gradient eluation 0.001 to 0.2 molar). The pure fraction is freeze-dried several times. It is possible in the same manner, that is using the same synthesis sequence, to produce the Asp(NH -eledoisin and the Asp(NH -Val -eledoisin.

EXAMPLE 2 (a) Carbobenzoxy L asparaginyl L alanyl L phenylalanine-methyl ester 19.3 g. of carbobenzoXy-L-asparagine-p-nitrbphenylester are dissolved in dimethyl formamide and mixed with L-alanyl-L-phenylalanine methyl ester (obtained from 17.1 g. of the hydrochloride with 8.5 cc. of triethylamine). After standing for 2 days at room temperature, the reaction product is filtered off under suction and washed with ether. The yield is 22.6 g. The melting point is 209-211 C. [oc] =15.1 (c.:l, glacial acetic acid).

.(b) L-asparaginyl-L-alanyl-L-phenylalanine-methyl-ester hydrochloride 15 g. of the carbobenzoxy compound are dissolved in 330 cc. of methanol/glacial acetic acid/1 n HCl 5:5 :1 and hydrogenated in the presence of palladium black. The yield is 10.0 g. The melting point is l68172 C. [a] =l6.l (c.=l, glacial acetic acid).

(c) L pyroglutamyl-L-prlyl-L-seryl-etert.-tubyloxycarbonyl L-lysyl-L-asparaginyl-L-alanyl-L-pheny[alaninemethyl ester 5.4 g. of L-pyroglutamyl-L-prolyl-L-seryl-e-terL-butyloxycarbonyl-L-lysine and L-asparaginyl-L-al-anyl-L-phenylalanine-methyl ester (obtained from 4.0 g. of the hydrochloride by reaction with 1.4 cc. of triethylamine) are coupled to the heptapeptide in the presence of 2.5 g. of dicyclohexylcarbodiimide. It is treated in aqueous methanol with a basic exchanger and an acid exchanger to remove the polar portion. The yield is g.

The same compound can be obtained in the following manner:

(d) Carbobenzoxy e tert.-butyloxycarbonyl-L-lysyl-L- asparaginyl-L-alanyl-L-phenylalanine-methyl ester (e) Cal-bobenzoxy L prolyl-L-seryl-e-tert.-butyl xycarbonyl L asparaginyl-L-alanyl-L-phenylalanine-methyl ester 7.3 g. of the carbobenzoxy compound obtained according to Example 2d are hydrogenated in 130 cc. of dimethyl torm amide in the presence of palladium black and the solution, after filtration off of the catalyst, is mixed with carbobenzoxy-L-prolyl-L-serinazide (obtained from 2.7 g. of the hydrazide by the reaction in 20 cc. of a 1.5 n solution of hydrogen chloride in tetrahydrofurane with 1.4 cc. of tert.-butylnitrite). The yield is 6.5 g. The melting point is 199-201 C.

(f) L pyroglutamyl L prolyl-L-seryl-e-tert.-butyl0xycarbonyl L lysyl L-asparaginyl-L-alanyl-L-phenylalanine-methyl ester The compound obtained under Example 2e is subjected to catalytic hydrogenation as above described and mixed with carbobenzoxy-L-pyroglutamic acid-p-nitrophenyl ester. After renewed hydrogenation, the product which is obtained is identical to that obtained under Example 20.

(g) L pyroglutamyl L prolyl-L-seryl-e tert.-butyloxycarbonyl L lysyl -L asparaginyl-L-alanyl-L-phenylalanine The above compound is obtained from the methyl ester (Example 2c and 2 'by enzymatic saponification as described in Example 1e. For further working up, the en- Zyme is denatured by warming for a short time, filtered under suction and the solution is freeze-dried. The yield is 90%.

885 mg. of L pyroglutamyl L prolyl L seryle tert. butyloxycarbonyl L lysyl L asparaginyl- L-alanyl-L-phenylalanine are reacted with 690 mg. of L- isoleucyl glycyl L leucyl L methioninamide according to the carbodiim-ide method in dimethyl formamide. After standing for 2 days at 0 C. an additional 2 days at room temperature, the precipitated dicylohexyl urea is filtered off under suction, the remaining liquid is concentrated and the thus obtained crude product is washed with 10% citric acid solution, water, saturated sodium bicarbonate solution and water. The yield is 650 mg. The melting point is 207209 C.

For splitting otf of the e-tert.-butyloxycarbonyl group from the lysine, 180 mg. are dissolved in 3 cc. of trifiuoro acetic acid and permitted to stand at room temperature for 45 minutes. After precipitation with ether, the product is subjected to chromatography over a SE-Sephex column (ammonium acetate-gradient pH 5.5, 0.001 to 0.2 molar).

EXAMPLE 3 L pyroglutamyl L prolyl L seryl L lysyl L asparaginyl L alanyl L phenylalanyl-L-valyl-glycyl- L-leucyl-L-methi0ninamide This compound is produced in analogous manner from L pyroglutamyl L prolyl L seryl e tert.-butyloxycarbonyl L lysyl L asparaginyl L alanyl L- phenylalanine and L-valyl-glycyl-L-leucyl-L-methioninamide.

EXAMPLE 4 L pyroglutamyl L prolyl L seryl e tert.- butyloxycarbonyl L lysyl L asparagyl [3 tert.- butylester L alanyl L phenylalanine-methyl ester [M.P.=l55 C., [a] =6l.0 C. (c.=l, glacial acetic acid)] is produced in analogous manner starting from carbobenzoxy L asparagyl ,8 tert.-butylester- L alanyl L phenylalanine-methyl ester [obtained from carbobenzoxy-L-asparagine acid-,B-terL-butylester and L- alanyl-L-phenylalanine-methyl ester, M.P.=114115 C., [oc] =22.2 (c.=1, ethanol)] by catalytic hydrogenation and coupling with L-pyroglutamyl-L-prolyl-L-seryl-etert.-butyloxycarbonyl-L-lysine. After enzymatic saponification, coupling with Lleucyl-glycyl-L-leucylL-methioninamide and normal splitting off of the protective groups and purification, the resulting compound is Leu -eledoisin.

EXAMPLE 5 T ert.-butyl0xycarb0nyl-glycyl-L-alanyl-L- phenylalanine-methyl ester 17.5 g. of tert.-butyloxycarbonyl-glycine is converted into the mixed anhydride at -15 C. in 150 cc. of dimethyl formamide with 14 cc. of triethylamine and 10 cc. of chloroformic acid ethyl ester and mixed with a solution of 31.5 g. of H-L-alanyl-L-phenylalanine-methyl ester-hydrochloride and 15.4 cc. of triethylamine in 150 cc. of dimethyl formamide. After working up in usual manner the above given compound is obtained in a yield of 31.5 g. (77%). The melting point is 106l08 C.; [oc] =-24.6 (c.=1, methanol).

(b) Tert.-butyloxycarbonyl-glycyl-L-alanyl-L- phenylalanine-lzydrazide The above compound is obtained from the methyl ester by reaction with four times the amount of hydrazine hydrate in methanol. The yield is 89%. The melting point is 177183 C.; [a] =27.7 (c.=1, glacial acetic acid).

(0) T ert. butyloxycarbonyl glycyl L-alanyl-L-phenyl alanyl-L-isoleucyl-glycyl-L-leucyl-L-methioninamide 1.22 g. of tert. butyloxycarbonyl glycyl L alanyl- L-phenylalaninehydrazide are suspended at -20 C. in

3.6 cc. of 2.2 n solution of hydrochloric acid in tetrahydrofurane and converted into the azide with 0.37 cc. of tert.-butylnitrite. After the reaction with 40 cc. of cold ethyl ester it is washed with a saturated sodium bicar- (d) H glycyl L alanyl L phenyIalanyl-L-isleucylglycyl L leucyl L methioninamide hydrochloride 1 .SH O

The above compound is obtained from 2 g. of the tert.- butyloxycarbonyl compound by splitting off of the protective groups with hydrochloric acid in glacial acetic acid in the above described manner. The yield is 1.6 g. (88%). The melting point is 240-260" C.; [u] =-45.8 (c.=1, glacial acetic acid).

(e) L pyroglutamyl L prolyl L seryl L lysylglycyl L alanyl L phenylalanyl L isoleucylglycyl-L-leucyl-L-methioninamide 1.6 g. of the heptapeptide of Example a are mixed in dimethyl formarnide with 0.3 cc. of triethylamine and after filtering off the triethyl ammonium chloride it is further reacted with 1.2 g. of Lpyroglutamyl-L-prolyl- L-seryl-e-tert.-butyloxycarbonyl-L-lysine and 4.4 g. of dicyclohexyl carbodiimide. After standing for 3 days at 0 C. and after destruction of the excess carbodiimide with a small amount of 2 n acetic acid, it is subjected to suction filtration to remove the precipitated dicyclohexyl urea, the filtrate is concentrated and in normal manner rubbed with citric acid solution, water, saturated sodium bicarbonate solution and water, and then dried. The yield is 1.8 g. The melting point is 223-225 C. By splitting off the protective groups in normal manner with trifluoro acetic acid and purification by chromatography on an SE-Sephadex, as described in Example lg, there is obtained the Gly eledoisin.

EXAMPLE 6 The compound Gly -Leu eledoisin is obtained in analogous manner from tert.-butyloxycarbonyl-glycyl-L- alanyl-L-phenylalanyl L leucyl L glycyl L-leucyl-L- methioninamide [by the reaction of tert.-butyloxycarbonyl-glycyl-L-alanyl-L-phenylalanine hydrazide and H-L-leucyl-glycyl-L-leucyl-L-methioninamide; M.P.=227- 229 C., [a] =32.6 (c.=1, dimethyl forn1amide)], splitting off of the protective groups and coupling with L-pyroglutamyl-L-prolyl L seryl-e-tert.-butyloxycarbonylL-lysine.

10 EXAMPLE 7 Gly -eledoisin and Gly -Leu -eledoisin can be prepared in accordance with the synthesis of Table 1, analogously to Examples 1-4, from glycyl-L-alanyl-L-phenylalaninemethyl ester-hydrochloride [obtained from the tert.- butyloxycarbonyl compound of Example 511 by splitting off of the protective groups, M.P.=206-210 C., [a] =13.5 (c.=1; methanol)], reaction with L-pyroglutamyl-L-prolyl L seryl e tert.-butyloxycarbonyl-L- lysine according to the carbodiimide method, enzymatic saponification of the reaction product and renewed carbodiimide coupling with L-isoleucyl-glycyl-L-1eucyl-L- methioninamide or L-leucyl-glycyl-L leucyl L methioninamide, respectively.

Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can by applying current knowledge readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this invention and, therefore, such adaptations should and are intended to be comprehended within the meaning and range of equivalence of the following claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. An undecapeptide of the formula:

L-pyroglutamyl-L-prolyl-L-seryl-L-lysyl-R -L-alanyl-L- phenylalanyl-R -glycyl-L-leucyl-L-rnethioninamide References Cited by the Examiner UNITED STATES PATENTS 6/1955 Block et al. 260-1125 1/ l 7 Nishizawa 16765 9/195-8 Brenner 260-112 2,866,783 12/1958 Bovarnick 260-112 3,153,613 10/1964 Jones et al. 16765 LEWIS GOTTS, Primary Examiner.

JULIAN S. LEVITT, Examiner.

MARTIN J. COHEN, PERRY A. STITH,

Assistant Examiners. 

1. AN UNDECAPEPTIDE OF THE FORMULA: 